Methods and means for producing barley yellow dwarf virus resistant cereal plants

ABSTRACT

The invention relates to methods for producing transgenic cereal plants resistant to Barley Yellow Dwarf Virus, particularly in the presence of co-infecting Cereal Yellow Dwarf Virus, by stably integrating into the cells of the transgenic plant a chimeric gene comprising a DNA region operably linked to plant expressible promoter in such a way that the RNA molecule may be transcribed from the DNA region, the RNA molecule comprising both sense and antisense RNA capable of pairing and forming a double stranded RNA molecule or hairpin RNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. ProvisionalApplication Serial No. 60/244,209, filed Oct. 31, 2000, the contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods for producing transgenic cereal plantsresistant to Barley Yellow Dwarf Virus, particularly in the presence ofco-infecting Cereal Yellow Dwarf Virus, by stably integrating into thecells of the transgenic plant a chimeric gene comprising a DNA regionoperably linked to plant expressible promoter in such a way that a RNAmolecule may be transcribed from the DNA region, the RNA moleculecomprising both sense and antisense RNA capable of pairing and forming adouble stranded RNA molecule or hairpin RNA.

DESCRIPTION OF RELATED ART

Barley yellow dwarf virus-PAV (BYDV-PAV) is the most serious andwidespread virus of cereals worldwide. The barley yellow dwarf virus(BYDV), also called red leaf in oats, can infect barley, oats, rye andwheat as well as numerous species of grasses. It occurs in most parts ofthe world and is considered the most common viral disease of cerealcrops.

BYDV (and CYDV) have been reported to cause cereal disease in over 50countries. In almost all cases where the species has been determined,the major losses have been due to BYDV-PAV (Barker & Waterhouse, 1999).Lost production due to BYDV averages about 15% in barley, 17% in wheatand 25% in oats (Lister and Ranieri, 1995). BYDV infection can affectplant height, grain size and grain quality. For example, BYDV infectionof barley can reduce the grain quality such that it is suitable only foranimal feed rather than malting.

BYDV is transmitted by several species of aphids. As the aphids (wingedor wingless) feed on the cereal crop, they transmit the virus throughtheir mouthparts. The aphids can remain infectious for life, which isaround 40 days.

Disease symptoms vary with the host species and the stage of cropdevelopment. Infections at the seedling stage may result in death ordwarfing as well as sterile heads. Leaves turn yellow from the tip down,along the leaf margins or in blotchy patches. Infected barley leaves,particularly flag leaves, turn bright yellow. In oats, the leaves mayturn from red to purple. Discolored areas enlarge and progress to thebase of the plant. Heads may be wholly or partially sterile. There mayalso be an increase or decrease of tillers produced by infected plants.Cereal plants infected early in the season may be shaded out by healthyor late infected surrounding plants. Winter wheat seedlings may be 100percent infected with BYDV before freeze-up in the fall. BYDV affectsyields by stunting, reduced tillering, sterility, and failure to fillkernels.

Natural resistance genes against this luteovirus give inadequatecontrol. Sources of natural resistance to BYDV and CYDV are rare (forreviews see Barker and Waterhouse 1999; Burnett et al., 1995). Inbarley, the Yd2 gene (Paltridge et al., 1998), originally identified inEthiopian concessions (Rasmussin and Schaller, 1959; Schaller et al.,1964), can confer resistance against BYDV-PAV, but its effectivenessvaries depending on the genetic background of the plant and growthconditions (Schaller, 1984; Larkin et al., 1991). The Bdv1 gene conferssome tolerance to BYDV (Singh et al, 1993) and has been introduced intosome wheat cultivars, such as Anza. However, BYDV replicates and causessymptoms and yield loss in plants containing either the Yd2 or Bdv1genes.

Previous attempts to introduce synthetic resistance into cereals haveproduced variable results. Virus resistance in plants containingvirus-derived transgenes, usually by the expression of functional ordysfunctional coat protein, movement or polymerase genes, has beenwidely reported (for review see Waterhouse & Upadhyaya 1998) andattempts have been made to produce transgenic plants with resistance toa few different luteoviruses, by expression of coat protein andpolymerase genes.

McGrath et al. (1997) transformed oat and barley plants with transgenesderived from the coat protein genes of BYDV-PAV and CYDV-RPV andobtained some resistant plants. However, this resistance was not stable.

In another study, Koev et al. (1998) transformed oat plants with the5′half of the BYDV-PAV genome and found one line that after inoculationwith BYDV-PAV showed disease symptoms but recovered and produced seed.Although BYDV resistance in the progeny of this line was inherited, thelevels of resistance varied greatly among individual plants, rangingfrom substantial to undetectable.

Waterhouse et al., 1998, Wang & Waterhouse, 2000 and Smith et al., 2000describe that virus immunity and posttranscriptional gene silencing(PTGS) can be induced in plants using transgenes that encode doublestranded (ds) or self-complementary “hairpin” (hp) RNA.

WO 98/53083 describes constructs and methods for enhancing theinhibition of a target gene within an organism involve inserting intothe gene-silencing vector an inverted repeat sequence for all or part ofa polynucleotide region within the vector. The inverted repeat sequencemay be a synthetic polynucleotide sequence or comprise a modifiednatural phenotype.

WO 99/32619 provides a process of introducing RNA into a living cell toinhibit gene expression of a target gene in that cell. The RNA has aregion with a double-stranded structure. Inhibition is sequence-specificin that the nucleotide sequence of the duplex region of the RNA and of aportion of the target gene are identical.

WO 99/49029 relates to a method of modifying gene expression and tosynthetic genes for modifying endogenous gene expression in a cell,tissue or organ of a transgenic organism, in particular a transgenicanimal or plant. Recombinant DNA technology is used topost-transcriptionally modify or modulate the expression of a targetgene in a cell, tissue, organ or whole organism, thereby producing novelphenotypes. Synthetic genes and genetic constructs which are capable ofrepressing, delaying or otherwise reducing the expression of anendogenous gene or a target gene in an organism when introduced theretoare also provided.

WO 99/53050 provides methods and means for reducing the phenotypicexpression of a nucleic acid of interest in eukaryotic cells,particularly in plant cells, by introducing chimeric genes encodingsense and antisense RNA molecules directed towards the target nucleicacid, which are capable of forming a double stranded RNA region bybase-pairing between the regions with sense and antisense nucleotidesequence or by introducing the RNA molecules themselves. Preferably, theRNA molecules comprise simultaneously both sense and antisensenucleotide sequence.

WO 99/61631 relates to methods to alter the expression of a target genein a plant using sense and antisense RNA fragments of the gene. Thesense and antisense RNA fragments are capable of pairing and forming adouble-stranded RNA molecule, thereby altering the expression of thegene. This publication also relates to plants, their progeny and seedsderived thereof, obtained using the methods described.

WO 00/49035 discloses a method for silencing the expression of anendogenous gene in a cell, the method involving overexpressing in thecell a nucleic acid molecule of the endogenous gene, wherein theoverexpression of the nucleic acid molecule of the endogenous gene andthe antisense molecule in the cell silences the expression of theendogenous gene.

SUMMARY AND OBJECTS OF THE INVENTION

The invention provides DNA molecules comprising a plant-expressiblepromoter operably linked to a DNA region which when transcribed in thecells of a cereal plant yields an RNA molecule comprising a firstnucleotide sequence of at least 19 bp having at least 70% nucleotidesequence identity to the sense nucleotide sequence of a BYDV isolateencoding the polymerase gene and a second nucleotide sequence of atleast 19 bp having at least 70% nucleotide sequence identity to thecomplement of the sense nucleotide sequence of an RNA dependent RNApolymerase (hereinafter referred to as “polymerase”) gene of a BYDVisolate, such as but not limited to a polymerase comprising thenucleotide sequence of SEQ ID No 1 from the nucleotide at position 1 tothe nucleotide at position 1014, and optionally a transcriptiontermination and polyadenylation signal, wherein the first and secondnucleotide sequence are capable of forming a double stranded RNA by basepairing between regions which are complementary. The first nucleotidesequence may comprise the nucleotide sequence of SEQ ID No 1 and thesecond nucleotide sequence may comprise the complement of the nucleotidesequence of SEQ ID No 1. The DNA molecules of the invention may containa nucleotide sequence encoding a spacer region between the DNA regionencoding the first nucleotide sequence and the DNA region encoding thesecond nucleotide sequence. The spacer region may have the nucleotidesequence of SEQ ID No 2.

The invention also provides a method for producing a cereal plant, suchas a cereal plant selected from the group of wheat, barley, rye andoats, e.g. barley, resistant to a Barley Yellow Dwarf Virus comprisingthe steps of producing a population of transgenic cereal plant linescomprising the DNA molecules of the invention integrated into the genomeof the cells of transgenic plant of the plant line and selecting atransgenic cereal plant line resistant to Barley Yellow Dwarf virusinfection and optionally comprising the further step of crossing theselected transgenic cereal plant line resistant to Barley Yellow Dwarfvirus infection to another cereal plant to obtain progeny plantscomprising the DNA molecules of the invention.

Another objective of the invention is to provide a method for producinga cereal plant resistant to a Barley Yellow Dwarf Virus in the presenceof a post transcriptional gene silencing inactivating protein, which maybe encoded by a co-infecting virus, particularly Cereal Yellow DwarfVirus, comprising the step of producing a population of transgeniccereal plant lines comprising a DNA molecule of the invention integratedinto the genome of the cells of transgenic plant of said plant line andselecting transgenic cereal plant lines resistant to Barley Yellow DwarfVirus infection.

Yet another objective of the invention is to provide use of a DNAmolecule of the invention to produce a transgenic cereal plant resistantto BYDV.

It is also a further objective of the invention to provide the use of aDNA molecule of the invention to produce a transgenic cereal plantresistant to BYDV in the presence of Cereal Yellow Dwarf virus (CYDV).

The invention further provides cereal plants and plant lines, which maybe selected from the group consisting of wheat, barley, rye and oat,e.g. barley plant and plant lines comprising stably integrated into thegenome of the cells of the cereal plant a DNA molecule according to theinvention wherein the cereal plant is resistant to BYDV virus infectionand replication of said virus, also in the presence of CYDV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Genome map of BYDV-PAV showing regions used to generatehpBYDVpol. (B) Design of hpBYDVpol construct. (C) Diagram ofself-complementary (hairpin) RNA produced by hpBYDVpol. RB: rightborder; nos 3′: nopaline synthase 3′ region; 35S-P: cauliflower mosaicvirus 35S promoter; tm1′: tumor morphology large gene 3′ region; PAVpol:BYDV-PAV polymerase gene sequence; JGMV5′: Johnson grass mosaic virus 5′untranslated region; Ubi-P: maize polyubiquitin gene promoter; LB: leftborder.

FIG. 2. Southern blot analysis of 15 primary T₀ hpBYDVpol barleytransformants. DNA was digested with Hind III, separated byelectrophoresis, blotted to Hybond N+™ membrane and hybridized with aradioactively labeled hpt probe. The number of intense bands in each ineach lane should represent the number of transgene copies in the plantline. Weak bands may be due to partial digestion of DNA.

FIG. 3. Southern blot analysis of T1 progeny of hpBYDVpol Lines 2 and 4.DNA from two resistant (R) and one susceptible (S) T1 plants for Lines 2and 4, and for non-transgenic barley (C), was digested with Apa I orBamH I, separated by electrophoresis, blotted to Hybond N+™ membrane andhybridized with a radioactively-labeled hpt probe. The number of bandsin the Apa I lanes and the intensity of the 3.75 KB bands for the BamH Ilanes should indicate copy number.

FIG. 4. Relationship between virion accumulation and inheritance of thehpBYDVpol transgene. Virus levels detected by ELISA 21 (white bar), 28(gray bar) and 42 (black bar) days after inoculation in (A) 14 T₁progeny of Line 2 and (B) 12 T₁ progeny of Line 4. Displayed above eachhistogram is a symbol representing the severity of viral symptoms inmature plants and an agarose gel containing PCR products from thecorresponding plant samples; the presence of a 626 bp product indicatesthe amplification and detection of the hpBYDVpol transgene.

FIG. 5. Reaction of transgenic barley, non-transgenic barley, and oatsto BYDV and CYDV. A. Reaction of transgenic, and non-transgenic barleyplants to BYDV-PAV. Two T1 hpBYDVpol resistant plants of Line 2 (left)and two non-transformed barley plants (right). B. Coast-black oats(Avena sativa) infected with BYDV-PAV, CYDV-RPV and B/CYDV-MIX, amixture of both isolates.

FIG. 6. BYDV-PAV and CYDV-RPV accumulation in a population ofsegregating progeny from hpBYDVpol Line 2. Virus levels, detected bystrain-specific ELISA, 21 days after inoculation, in 15 progeny plantsinoculated with B/CYDV-MIX (a complex of both BYDV-PAV and CYDV-RPV).The pair of dark and light histogram bars for each progeny plantrepresent the levels of BYDV-PAV and CYDV-RPV detected, respectively.The + and − above each pair indicate whether or not the plant hasinherited the hpBYDVpol transgene. The transgene status was determinedby PCR as in FIG. 3. C+ and C− are results for non-transgenic controlplants that have been challenged and not challenged with B/CYDV-MIX,respectively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Luteoviruses, such as Barley Yellow Dwarf Virus (“BYDV”) have been amongthe most recalcitrant viral group to transgene-mediated resistance(Barker and Waterhouse, 1999). Despite the economic importance of thecereal diseases caused by BYDV, previous attempts to produce transgeniccereals with protection against BYDV have been unsuccessful.

While some attempts (McGrath et al., 1997; Koev et al., 1998) haveproduced oat or barley plants with resistance (reduced virusreplication) or tolerance (reduced virus symptoms but unimpeded virusreplication) to BYDV, the inheritance of the resistance/tolerance hasbeen variable. This inheritance has been further complicated by thecomplex transgene insertion patterns in such plants, especially thoseobtained using biolistic transformation.

The prior art is thus deficient in providing methods and means forobtaining BYDV resistant cereal plants, with stable inheritance of theresistance gene, using a transgenic approach. The current invention hassolved these and other problems as set forth hereinafter in the variousembodiments, as well as in the claims.

The current invention is based on the finding that transgenic cerealplants, such as barley, could be made extremely resistant to BYDVinfection, replication and disease symptom development using a chimericgene comprising a promoter which could be expressed in cereal plantcell, and could drive the transcription of an operably linked DNAregion, the transcription resulting in an RNA molecule comprising bothsense and antisense parts of a BYDV genome corresponding to thenucleotide sequence encoding the RNA dependent RNA polymerase (ORF1).The sense and antisense parts are essentially complementary to eachother and thus capable of forming a double stranded RNA. For the virusresistance to occur such a chimeric gene need only to be present as asingle transgene, thus resulting in inheritance in a simple Mendelianmanner.

In addition, it was realized that the BYDV disease resistance conferredby the chimeric genes according to the invention, could be observed evenin the presence of PTGS inactivating protein, such as may be found inthe co-infecting Cereal Yellow Dwarf Virus (CYDV-RPV).

Until recently, BYDV was described as having at least six differentserotypes, which fell into two different subgroups (Waterhouse et al.,1988; Martin and D'Arcy, 1995). However, comparison of their nucleotidesequences has led to redefining PAV and MAV as species of BYDV, and RPVas a species of Cereal yellow dwarf virus (CYDV; Mayo & D'Arcy 1999;Wang et al., 1998). The 3′ halves of the two viruses encode movement andcoat protein genes and share a fair degree of homology. Their 5′ halvesencode polymerase gene sequences, which are more closely related tothose of other groups of viruses than to each other (Miller et al.,1995; Wang et al., 1998a).

In one embodiment of the invention, a method is provided for producing acereal plant or plant line resistant to Barley Yellow Dwarf virusinfection, virus replication and disease symptom development comprisingthe steps of

a) producing a population of transgenic cereal plant lines comprising aDNA molecule integrated into the genome of the cells of a transgenicplant whereby the DNA molecule comprises the following operably linkedelements:

(1) a plant-expressible promoter;

(2) a DNA region which when transcribed in the cells of a cereal plantyields a RNA molecule comprising

(a) a first nucleotide sequence of at least 19 bp having at least 70%nucleotide sequence identity to the sense nucleotide sequence of a BYDVisolate encoding RNA dependent RNA polymerase; and

(b) a second nucleotide sequence of at least 19 bp having at least 70%nucleotide sequence identity to the complement of said sense nucleotidesequence of a BYDV isolate encoding RNA dependent RNA polymerase; andoptionally

(c) a transcription termination and polyadenylation signal;

wherein the first and second nucleotide sequence are capable of forminga double stranded RNA by base pairing between regions which arecomplementary; and

b) isolating transgenic cereal plants or plant lines resistant to BarleyYellow Dwarf Virus infection.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970) The computer-assisted sequence alignment above, can beconveniently performed using standard software program such as GAP whichis part of the Wisconsin Package Version 10.1 (Genetics Computer Group,Madison, Wis., USA) using the default scoring matrix with a gap creationpenalty of 50 and a gap extension penalty of 3.

For the purpose of the invention, the “complement of a nucleotidesequence represented in SEQ ID No: X” is the nucleotide sequence whichwould be capable of forming a double stranded DNA molecule with therepresented nucleotide sequence, and which can be derived from therepresented nucleotide sequence by replacing the nucleotides throughtheir complementary nucleotide according to Chargaff's rules (AT; GC)and reading in the 5′ to 3′ direction, i.e. in opposite direction of therepresented nucleotide sequence.

As used herein, nucleotide sequences of RNA molecules may be identifiedby reference to DNA nucleotide sequences of the sequence listing.However, the person skilled in the art will understand whether RNA orDNA is meant depending on the context. Furthermore, the nucleotidesequence is identical except that the T-base is replaced by uracil (U)in RNA molecules.

The length of the first and second nucleotide sequences may vary fromabout 10 nucleotides (nt) up to a length equaling the length innucleotides of part of the BYDV genome encompassing the RNA dependentRNA polymerase encoding ORF of a BYDV genome (normally referred to asORF 1). The length of the first or second nucleotide sequence may be atleast 15 nt, or at least about 20 nt, or at least about 50 nt, or atleast about 100 nt, or at least about 150 nt, or at least about 200 nt,or at least about 500 nt, or at least about 1600 bp.

It will be appreciated that the longer the total length of the firstnucleotide sequence is, the less stringent the requirements for sequenceidentity between the total sense nucleotide sequence and thecorresponding sequence in the target gene become. The total firstnucleotide sequence can have a sequence identity of at least about 75%with the corresponding target sequence, but higher sequence identity canalso be used such as at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, about 100%. The first nucleotide sequencecan also be identical to the corresponding part of BYDV genome. However,it is advised that the first nucleotide sequence always includes asequence of about 19 or 20 consecutive nt, or even of about 50consecutive nt, or about consecutive 100 nt, or about 150 consecutive ntwith 100% sequence identity to the corresponding part of BYDV genome.For calculating the sequence identity and designing the correspondingfirst nucleotide sequence, the number of gaps should be minimized,particularly for the shorter sense sequences.

The length of the second (antisense) nucleotide sequence is largelydetermined by the length of the first (sense) nucleotide sequence, andmay correspond to the length of the latter sequence. However, it ispossible to use an antisense sequence that differs in length by about10% without any difficulties. Similarly, the nucleotide sequence of theantisense region is largely determined by the nucleotide sequence of thesense region, and may be identical to the complement of the nucleotidesequence of the sense region. Particularly with longer antisenseregions, it is however possible to use antisense sequences with lowersequence identity to the complement of the sense nucleotide sequence,such as at least about 75% sequence identity, or least about 80%, or atleast about 85%, more particularly with at least about 90% sequenceidentity, or at least about 95% sequence to the complement of the sensenucleotide sequence. Nevertheless, it is advised that the antisensenucleotide sequences always includes a sequence of about 19 or 20consecutive nucleotides, although longer stretches of consecutivenucleotides such as about 50 nt, or about 100 nt, or about 150 nt with100% sequence identity to the complement of a corresponding part of thesense nucleotide sequence may be used. It is clear that the length ofthe stretch of the consecutive nucleotides with 100% sequence identityto the complement of the sense nucleotide sequence cannot be longer thanthe sense nucleotide sequence itself. Again, the number of gaps shouldbe minimized, particularly for the shorter antisense sequences. Further,it is also advised that the antisense sequence has between about 75% to100% sequence identity with the complement of the 5′ half of the BYDVgenome.

In one embodiment of the invention, the DNA molecules according to theinvention may comprise a DNA region encoding a spacer between the DNAregion encoding the first and second nucleotide sequences. As indicatedin WO 99/53050 the spacer may contain an intron to enhance the BYDVresistance. In one embodiment thereof, the spacer may comprise thenucleotide sequence of SEQ ID No 2.

In another embodiment of the invention, the used DNA molecule has afirst and second nucleotide sequence comprising a nucleotide sequence ofat least 19 or 20 bp having at least 75% sequence identity to anucleotide region of a BYDV genome encompassing an ORF comprising thenucleotide sequence of SEQ ID No 1 from the nucleotide at position I tothe nucleotide at position 1014, or its complement. It goes withoutsaying that again the sequence identity may be higher, as indicatedelsewhere in the specification or the length of the comprised nucleotidesequence may be higher, again as indicated elsewhere in thisspecification.

In another embodiment of the invention, the first nucleotide sequencecomprises the nucleotide sequence of SEQ ID No 1 and the secondnucleotide sequence comprises the nucleotide sequence of the complementof SEQ ID No 1.

However, the nucleotide sequence of the complete genome of a number ofBYDV isolates is known and available through databases such as, e.g.,Genbank, and the person skilled in the art can easily identify thenucleotide sequences corresponding to the sequence of SEQ ID No 1 inthose nucleotide sequences. These nucleotide sequences derived fromother BYDV isolates can be used to generate BYDV resistant cereal plantsand plant lines to equal effect.

The following nucleotide sequences of complete genomes of BYDV isolatesare available: Genbank accession Nr NC_(—)002160/GI:9634102; Genbankaccession Nr NC_(—)001599/GI:9627413; Genbank accession NrAF235167/GI:7417288; Genbank accession Nr AF218798/GI:6715477; GenbankAccession Nr D85783/GI:1395150; GenbankAccession Nr D11032/GI:221098;GenbankAccession Nr D10206/GI:221091; Genbank Accession NrD11028/GI:221084; Genbank Accession Nr X07653/GI:58798; GenbankAccession Nr L25299/GI:408929.

Sequences corresponding to the nucleotide sequence used to exemplify thecurrent invention may be identified in other BYDV genomes e.g. by usingcomputer-assisted alignment programs. Alternatively, the sequences canbe identified and isolated using PCR and the oligonucleotide primer withnucleotide sequences as in SEQ ID 3 and 4, essentially similarnucleotide sequences (having at least 85% sequence identity, or at least90% sequence identity).

It will be clear that also nucleotide molecules hybridizing understringent conditions to a nucleotide comprising the sequence of SEQ IDNo 1 or similar corresponding sequences from other BYDV genomes may alsobe used to similar effect.

“Stringent hybridization conditions” as used herein mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% sequence identity between the probe and thetarget sequence. Examples of stringent hybridization conditions areovernight incubation in a solution comprising 50% formamide, 5× SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared carrier DNA such as salmon sperm DNA, followed by washing thehybridization support in 0.1× SSC at approximately 65° C. Otherhybridization and wash conditions are well known and are exemplified inSambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor, N.Y. (1989), particularly chapter 11.

The invention is also directed to a method of protecting a cereal plantagainst BYDV virus in the presence of a PTGS inactivating protein. Suchproteins and nucleotide regions encoding them have been identified inseveral viruses and endogenous orthologous genes in plants have beenidentified. As used herein, a PTGS inactivating protein is a protein,which when present in a plant cell results in a reduction of the posttranscriptional gene silencing observed when sense or antisense RNAmolecules are present in that cell.

In one embodiment, the PTGS inactivating protein is encoded by a virus,such as Cereal Yellow Dwarf Virus. The methods of the invention thereforcan also be used to produce cereal plants resistant to Barley YellowDwarf Virus, and Cereal Yellow Dwarf Virus co-infection.

It goes without saying that the DNA molecules for use in the inventionmay comprise a first nucleotide sequence comprising several differentsense nucleotide sequences corresponding to parts of a BYDV genome (ashereinbefore described) and a second nucleotide sequence comprising theseveral corresponding complementary nucleotide sequences of the severaldifferent sense nucleotide sequences in the first nucleotide sequence.Moreover, a single DNA molecule for use in the invention may comprisewithin its first nucleotide sequence simultaneously several differentsense nucleotide sequences corresponding to parts of a BYDV genome aswell as different sense nucleotide sequences corresponding to parts ofanother viral genome such as a CYDV genome.

Thus the invention also provides a method for protecting cereal plants,such as but not limited to barley plants, against a BYDV and CYDVco-infection, the method comprising

a) producing a population of transgenic cereal plant lines comprising aDNA molecule, wherein the DNA molecule comprises the following operablylinked DNA elements

i) a plant-expressible promoter;

ii) a DNA region which when transcribed in the cells of a cereal plantyields an RNA molecule comprising

1) a first nucleotide sequence comprising a multitude of nucleotidesequences, each comprising at least 19 or 20 bp having at least 70%nucleotide sequence identity to the sense nucleotide sequence of a BYDVisolate or a CYDV isolate

2) a second nucleotide sequence having at least 70% nucleotide sequenceidentity to the complement of the first nucleotide sequence; and

iii) a transcription termination and polyadenylation signal; wherein thefirst and second nucleotide sequence are capable of forming a doublestranded RNA by basepairing between the regions which are complementary;and

b) isolating transgenic cereal plant lines resistant to Barley YellowDwarf Virus and Cereal Yellow Dwarf Virus infection.

In one embodiment of the above mentioned method, the first nucleotidesequence comprises the nucleotide sequence of SEQ ID No 7 from thenucleotide at position 429 to the nucleotide at position 629(corresponding to ORF1 of BYDV Australian isolate with the genomicsequence available under Genbank Accession number X 07653) and thenucleotide sequence of SEQ ID No 7 from the nucleotide at position 2314to the nucleotide at position 2514 (corresponding to ORF2 of anAustralian BYDV isolate) and the nucleotide sequence of SEQ ID No 7 fromthe nucleotide at position 5052 to the nucleotide at position 5270(ORF6+3′ untranslated end of BYDV) and the nucleotide sequence of SEQ IDNo 8 and the nucleotide sequence of SEQ ID No 9 (the latter twosequences corresponding to part of a CYDV genome) and the secondnucleotide sequence comprises the about exact complement of the firstnucleotide sequence.

The invention also extends to DNA molecules for use to produce BYDVresistant cereal plants described herein as well as plant cells andcereal plants comprising a DNA molecule according to the invention.

The DNA molecules of the invention suitable for the production of BYDVor BYDV/CYDV resistant plants may be introduced into cereal plant lines,already comprising the natural BYDV resistance alleles such as thementioned Yd2 or Bdv1 genes to obtain an additive or synergistic effecton virus resistance.

The method and means of the invention are suited for cereal crop plantssuch as wheat, rye, oat and barley, but may also be used for protectingother grasses and small grain cereals susceptible to BYDV or closelyrelated viruses.

It will be clear that actual method of transforming the cereal crop haslittle importance for the current method and several methods includingAgrobacterium mediated transformation, microprojectile bombardment,electroporation of compact embryogenic calli, silicon whisker mediatedDNA introduction are available in the art.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the DNA molecule for obtaining BYDV resistance accordingto the invention in other varieties of the same or related plantspecies, or in hybrid plants. Seeds obtained from the transformed plantscontain the chimeric genes of the invention as a stable genomic insertand are also encompassed by the invention.

The following non-limiting Examples describe the construction of DNAmolecules suitable for use in making BYDV resistant cereal plant lines.Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrookand Russell (2001) Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA and in Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for plant molecular work are described in Plant MolecularBiology Labfax (1993) by R. D. D. Croy, jointly published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications,UK. Standard materials and methods for polymerase chain reactions can befound in Dieffenbach and Dveksler (1995) PCR Primer: A LaboratoryManual, Cold Spring Harbor Laboratory Press, and in McPherson at al.(2000) PCR-Basics: From Background to Bench, First Edition, SpringerVerlag, Germany.

All the nucleotide sequences identified in this text by their databaseaccession number are hereby incorporated by reference.

Throughout the description and Examples, reference is made to thefollowing sequences:

SEQ ID No 1: part of the Barley Yellow Dwarf Virus genome spanning ORF1and 5′ end of ORF2.

SEQ ID No 2: part of the Barley Yellow Dwarf Virus genome spanning the3′ end of ORF2.

SEQ ID No 3: PCR oligonucleotide primer 1

SEQ ID No 4: PCR oligonucleotide primer 2

SEQ ID No 5: PCR oligonucleotide primer 3

SEQ ID No 6: PCR oligonucleotide primer 4

SEQ ID No 7: nucleotide sequence of an Australian isolate of BYDV(Genbank accession X07653)

SEQ ID No 8: nucleotide sequence of part of an Australian isolate ofCYDV (from Genbank accession AF 020090)

SEQ ID No 9: nucleotide sequence of part of an Australian isolate ofCYDV (from Genbank accession AF 020090)

EXAMPLES

Experimental Procedures

Barley Transformation

Hordeum vulgare L. cv. ‘Golden Promise’ was transformed with thehpBYDVpol construct using the Agrobacterium inoculation procedure andtarget tissue as described by Tingay et al. (1997). Freshly dissectedscutella (with embryo axes removed) were immersed for 10 min in asuspension of Agrobacterium tumefaciens AGL1:hpBYDVpol orAGL0:hpBYDVpol, that was obtained by inoculating 5 mL of antibiotic-freeMG/L medium (Tingay et al., 1997) with 250 μL of overnight Agrobacteriumculture (grown in the presence of 50 mg/L spectinomycin and 25 mg/Lrifamipicin) with shaking at 28° C. The scutella were blotted brieflywith sterile filter paper to remove excessive liquid, placed with thesmooth side down on solid callus induction medium, and incubated at 25°C. for 2-3 days. The scutella were then transferred without washing tocallus induction medium containing 30 mg/L hygromycin and 150 mg/LTimentin™, again with the smooth side down. They were subcultured twice,at two weeks intervals, on callus induction medium containing 50 mg/Lhygromycin and 150 mg/L Timentin™ to produce hygromycin-resistant calli,which were subsequently transferred to regeneration medium containing 25mg/L hygromycin, and 150 mg/L Timentin™. Callus pieces forming shoots onthe regeneration medium were transferred either directly to rootingmedium containing 25 mg/L hygromycin or first to a half-strengthregeneration medium and then to rooting medium.

Testing Transgenic Plants for BYDV Resistance

Unless stated otherwise, aphids (Rhopalosiphum padi) were allowed tofeed for 48 hr on virus-infected oat leaves then transferred (5-10 perplant) onto ten-day-old test plants. The virus isolates used were anAustralian isolate of BYDV-PAV, an Australian isolate of CYDV-RPV and anAustralian B/CYDV-MIX isolate which contained both BYDV-PAV and CYDV-RPV(Waterhouse et al., 1986). After a 72 hr inoculation feeding period onthe test plants, the aphids were killed with pyrethrin and the plantstransferred to a glasshouse (16 hr day 18° C.; 8 hr night 13° C.).Virion accumulation was measured, unless stated otherwise, 21 and 28days after inoculation, by enzyme-linked immunosorbent assay (ELISA)(Xin et al., 1988), using monoclonal antibody diagnostic kits forBYDV-PAV and CYDV-RPV supplied by Sanofi Pasteur Diagnostics. Theconversion of substrate was measured after four hours incubation.

Analysis of Transgenic Plants by DNA Hybridization

Genomic DNA was isolated from barley leaves using the procedure ofLagudah et al. (1991). Approximately 10 μg DNA was digested overnightwith Hind III, Apa I or BamHI and electrophoresed in a 1% agarose gel(Sambrook et al., 1989). The DNA was blotted onto Hybond™-N+ positivelycharged nylon membrane using 20× SSC, in accordance with the proceduresupplied by Amersham Life Science. Hybridization was conducted at 42° C.in a formamide buffer using a radiolabeled probe from a 1.1 KB hpt(hygromycin resistance gene) fragment. The hybridized membranes werewashed as recommended in the Amersham protocol and analyzed using aPhosphorImager (Molecular Dynamics).

Analysis of Transgenic Plants by PCR

Genomic DNA for PCR template was prepared as for Southern analysis.Oligonucleotide primers (5′-TGTGGCAGTGGAGAGMGAG-3′ (SEQ ID No 5) and5′-ATGTTGTTGGTGATTTGGTG-3′ (SEQ ID No 6) were used to identify T1progeny containing the hpBYDVpol gene. These primers amplify a 626 ntfragment from ORF2 of BYDV. The amplified region forms part of thespacer loop between sense and antisense sequences of the hpBYDVpol gene.PCR analysis was performed using AmpliTaq Gold™ according tomanufacturers instructions using approximately 100 ng of genomic DNA.PCR reactions were performed in a thermal sequencer (FTS 960, Corbettresearch); 94° C. 12 min; 5 cycles 94° C. 1 min, 60° C. 2 min, 72° C. 3min; 25 cycles 94° C. 30 sec, 60° C. 2 min, 72° C. 1 min 30 sec; 70° C.1 min. Electrophoretic analysis of PCR products was conducted using 1%agarose gels buffered in 1× TBE.

Example 1 Construction of the Hairpin Gene (hpBYDVpol)

A full-length BYDV-polymerase (BYDVpol) sequence was amplified from acDNA clone of an Australian BYDV-PAV isolate (GenBank Accession No.D11032) using GeneAmp XL PCR kit (Perkin Elmer), with a pair of primers(5′-ACCATTCTATTGTGCTCTCGCACAGAGATAAGCAGGAAACCATGGTTTTCGA AATACTMTAGGT-3′(SEQ ID No 3) and 5′-CCGGAATTCTTAATATTCGTTTTGTGAG-3′ (SEQ ID No 4) thatintroduced a Nco I site and an EcoR I site at the 5′ end and the 3′ end,respectively. The underlined nucleotides are from the BYDV-PAV sequence.The resulting PCR fragment was digested with Nco I and EcoR I, andcloned into pUJJT (Wang and Waterhouse, 2000) at the correspondingsites, forming the Ubi1-JGMV5′-BYDVpol-tm1′ cassette. A 1.6 kb sequencefrom the 5′ half of BYDVpol was excised with Nco I and BamH I from thePCR fragment, treated with Klenow polymerase to generate blunt ends, andinserted into the EcoR I site (also pre-treated with Klenow) betweenBYDVpol and tm1′ in the Ubi1-JGMV5′-BYDVpol-tm1′ cassette, giving thehairpin gene hpBYDVpol (FIG. 1). For barley transformation, the hairpingene cassette was excised with Not I and inserted into pWBVec8, whichcontains an intron-interrupted hygromycin resistance gene, as theselectable marker, in the T-DNA region (Wang et al., 1998b).

Example 2 Transformation and Analysis of T₀ Plants

A gene construct (hpBYDVpol) was made in which a hairpin RNA, containingBYDV-PAV polymerase gene sequences, is transcribed under the control ofthe maize ubiquitin promoter (FIG. 1). Using this construct and anAgrobacterium-mediated transformation system, an overall transformationefficiency of 13% was achieved resulting in 38 independent transgenicbarley plants (Table 1).

TABLE 1 Transformation of barley with hpBYDVpol. Acetosyringone No. No.conc. in co- No. hygromycin transgenic Strain of cultivation scutellaresistant calli lines Agrobacterium medium (μM) used generated generatedAGL0 0 72  40 (55%)^(A) 17 (24%)  200 70 40 (57%) 8 (11%) AGL1 0 71 17(24%) 6 (8%)  200 72 22 (30%) 7 (10%) ^(A)Numbers in brackets = No.transgenic calli or plants/No. scutella (as a percentage)

Southern analysis indicated that 19 plants carried a single transgenecopy, 12 contained two copies and 7 had three or more copies. Theanalysis of the first 15 of these plants is shown in FIG. 2. When 25 ofthe T₀ plants were inoculated with BYDV-PAV, 9 of them appeared highlyresistant as they supported little or no virus replication when measuredby ELISA (data not shown). Six lines (1-6) were selected for furtheranalysis; four of these appeared highly resistant and two appearedsusceptible to BYDV-PAV infection (Table 2).

TABLE 2 Testing T₀ and T₁ hpBYDVpol plants for virus resistance. T₀ T₁Segregation T₀ T₀ Number of Highly resistant: Line Reaction to transgenecopies Susceptible number BYDV-PAV (Southern) (ELISA) 1 Highlyresistant >5  1:2 2 Highly resistant 1 18:12^(A) 3 Susceptible 1  0:15 4Highly resistant 1 17:10^(A) 5 Highly resistant 2 15:2^(B) 6Susceptible >5  0:30 ^(A)The segregation for BYDV resistance versussusceptibility conforms to a 3:1 ratio for a single dominant locus(Chi-square test, p > 0.05). ^(B)The segregation for BYDV resistanceversus susceptibility conforms to a 15:1 ratio for two dominant loci(Chi-square test, p > 0.05).

Example 3 Analysis of T₁ Plants for Response to BYDV-PAV Inoculation

About 20 seed from each of the 6 T₀ Lines were sown in soil. Theresulting plants were inoculated with BYDV-PAV and assayed by ELISAthree weeks later (Table 3).

TABLE 3 Virus level, measured by ELISA, for groupings of highlyresistant and susceptible T1 progeny of transgenic Lines 2 and 4,following inoculation with either BYDV-PAV or CYDV-RPV. Challenge virusCYDV-RPV Reaction to BYDV-PAV Highly infection Highly resistantSusceptible resistant Susceptible Line 2 0.103 ± 0.001^(A) (9)^(B) 1.148± 0.074 (6) −(0) 1.136 ± 0.061 (15) Line 4 0.109 ± 0.036 (9)  1.741 ±0.075 (3) −(0) 1.373 ± 0.107 (15) Control + 1.040 ± 0.172 (5)  1.070 ±0.102 (6) Control − 0.105 ± 0.003 (6)  0.147 ± 0.003 (6) ^(A)ELISAreading (with Standard Errors) of plants, 28 days post inoculation^(B)Numbers of plants are shown in brackets Control + = challengedwildtype plants; Control − = unchallenged wildtype plants

All of the plants from Lines 3 and 6 were as susceptible to BYDV-PAVinfection as their T₀ parents had been, whereas the progeny of each ofthe other lines (which were all resistant as T₀ plants) contained someplants that were susceptible and others that were resistant. The T₀ Line1 plant produced a total of eighteen seed and of these only threegerminated. One of the three plants that grew was found to be resistantto BYDV, while the other two were susceptible. The progeny of Lines 2and 4 conformed to a segregation ratio of 3:1 (highly resistant:susceptible), suggesting the presence of a single dominant transgenelocus in each line and Southern analysis (FIG. 3) revealed that the locieach appear to contain a single transgene. Plants from Line 5 conformedmost closely to a segregation ratio of 15:1 (highly resistant:susceptible), implying the presence of two dominant loci. Takentogether, the results suggested that the hpBYDVpol transgene wasconferring extreme resistance to BYDV-PAV in four of the six transgenicbarley lines analyzed and, in three of them (Lines 2, 4 and 5), this wasinherited in a predictable Mendelian manner.

Transgene inheritance and virus immunity in hpBYDVpol Lines 2 and 4Inheritance of the hpBYDVpol transgene and of BYDV-PAV resistance wasfurther examined in Lines 2 and 4. Fourteen T₁ plants of each line werechallenged with BYDV-PAV and monitored for virus symptoms and virionaccumulation 21, 28, and 42 days after inoculation. The inheritance ofthe hpBYDVpol transgene in these plants was examined by PCRamplification of a 626 bp fragment of DNA from the transgene. In bothlines (FIG. 4), the inheritance of hpBYDVpol correlated perfectly withlack of virus symptoms and resistance to BYDV infection. Plants thatwere symptomless and contained undetectable virus levels 21 days afterinoculation and remained this way throughout the six weeks of analysis(FIG. 5A), whereas those plants without the hpBYDVpol transgene showedvirus symptoms and virion accumulation at all three time-points. Theco-segregation of virus accumulation with the absence of the transgenewas also evident in the grain yield from the individual progeny plants.In Line 2, the average grain yield from the nine hpBYDVpol-containing T1progeny was 29.7±1.8 g compared to a yield of 20.9±1.5 g from the sixtransgene-free progeny. Twenty-eight days after inoculation, the ELISAlevel in plant 12 of Line 4 (FIG. 4) suggested that some virusaccumulation had occurred. However, the plant had undetectable levels ofBYDV-PAV both one week before and two weeks after this time-point, andnever developed virus symptoms. This suggests that there might have beena low level of virus replication, peaking at 28 days, which was thenovercome by the transgene-induced resistance or that there was somecontamination of the sample during analysis.

Specificity and Robustness of the Virus Resistance

Although BYDV-PAV is the major virus pathogen of cereals in the field,CYDV-RPV can also be present. Indeed the two viruses can occur as acomplex and appear to have a synergystic effect on symptom severity(FIG. 5B). The viruses share common hosts, a common insect vector andmany aspects of their biology. However, they are at opposite ends of theluteovirus spectrum in terms of sequence homology and genomeorganization. To test whether the hpBYDVpol transgene also conferredprotection against CYDV-RPV, approximately 15 T₁ progeny from each ofhpBYDVpol Lines 2 and 4 were challenged with BYDV-PAV and another 15 ofeach line with CYDV-RPV. Measuring the virus accumulation in theseplants by ELISA revealed that, as before, both lines segregated 3:1 forBYDV-PAV resistance. However, all of the plants inoculated with CYDV-RPVwere fully susceptible to the virus (Table 3). These results indicatethat the resistance conferred by the hpBYDVpol transgene is specific toBYDV-PAV. This might have been expected, as there is only 34% homologybetween nucleotide sequence in hpBYDVpol and the corresponding region inCYDV-RPV.

Some viruses have the capacity to enhance the replication and/or spreadof co-infecting viruses and to inactivate PTGS (Vance 1991; Vance etal., 1995; Pruss et al., 1997; Shi et al., 1997; Voinnet et al, 1999).Therefore, it was possible that infection by CYDV-RPV, which canco-infect with BYDV-PAV in the field, could enhance the replication andspread of BYDV-PAV or inactivate the hpBYDVpol-mediated BYDV-PAVimmunity, thus disarming the plant's newly conferred protection. It hasrecently been shown that some viruses have the capacity to inactivatePTGS (Voinnet et al. 1999). The P1 protein of the Sobemovirus, Riceyellow mottle virus, inactivates PTGS (Voinnet et al., 1999). Its genemay be orthologous to ORF1 (and possibly parts of ORF2) of CYDV-RPV.

Therefore, it was important to determine whether infection by CYDV-RPVcould inactivate the hpBYDVpol-mediated BYDV-PAV resistance. To testthis, 15 T₁ progeny from hpBYDVpol Line 4 were inoculated with theB/CYDV-MIX isolate, a virus complex containing BYDV-PAV and CYDV-RPV.The plants were subsequently tested using species-specific ELISA foraccumulation of BYDV-PAV and CYDV-RPV and by PCR for inheritance of thetransgene. The results (FIG. 6) showed that CYDV-RPV replicated to highlevels in all 15 plants but that the 12 plants inheriting the hpBYDVpoltransgene were resistant to BYDV-PAV.

This shows that the resistance to PAV is not compromised by replicationof CYDV-RPV and further confirms the 3:1 (BYDV-PAV resistance:susceptible) segregation ratio.

Recovery of Virus from Virus-Challenged hpBYDVpol Plants

Although BYDV-PAV-challenged hpBYDVpol-plants contain extremely lowlevels of BYDV-PAV antigen, they might contain sufficient virus to beacquired by aphids and thus be of ecological significance. To examinethis, we attempted to recover infectious virus from T1 progeny plantsfrom lines 2 and 4 that had been previously challenged with eitherBYDV-PAV or B/CYDV-MIX. Virus-free aphids were fed (for three days) onthe plants ten weeks after the initial challenge and then transferred tohealthy test plants (Table 4).

TABLE 4 Recovery of virus from transgenic and non-trausgenic barleyplants inoculated with BYDV-PAV or B/CYDV-MIX. Challenge Recovery ofRecovery of virus Genotype^(A) BYDV-PAV^(B) CYDV-RPV^(B) BYDV-PAV Line2 + t 0/4 (0.084 ± — 0.004)^(C) Line 2 − t 1/1 (2.069) — Line 4 + t 0/4(0.076 ± 0.003) — Line 4 − t 1/1 (1.185) — Control 3/3 (1.212 ± 0.081) —B/CYDV-MIX Line 2 + t 0/4 (0.069 ± 0.004) 4/4 (0.895 ± (BYDV-PAV 0.174)and CYDV- Line 2 − t 1/1 (1.191) 1/1 (0.819) RPV) Line 4 + t 0/4 (0.094± 0.025) 4/4 (1.576 ± 0.134) Line 4 − t 1/1 (0.954) 1/1 (0.909) Control3/3 (1.108 ± 0.166) 3/3 (1.473 ± 0.300) ^(A)+ t = segregant containingthe hpBYDVpol transgene; −t = segregant without hpBYDVpol ^(B)Number oftest plants infected/Number inoculated ^(C)Numbers in brackets areaverage ELISA readings and associated Standard Errors

Whereas the test plants became infected with BYDV-PAV from aphids fed onBYDV-PAV or B/CYDV-MIX challenged wildtype or non-transgene segregantplants, none of them was infected with BYDV-PAV from aphids fed onsimilarly challenged plants containing the hpBYDV-PAVpol transgene.However, aphids did recover CYDV-RPV from hpBYDVpol plants challengedwith the B/CYDV-MIX mixture. Taken altogether, the data show thatBYDV-PAV-challenged hpBYDVpol plants contain no biologically activevirus and should be regarded as immune to BYDV-PAV.

Example 4 Construction of a DNA Construct Encoding dsRNA with MultipleTarget Regions in the BYDV and CYDV Genomes

Using conventional recombinant DNA techniques, the following chimericgenes are constructed comprising the following operably linked DNAelements:

a maize ubiquitin promoter region (Christensen and Quail, 1996) orsubterranean clover stunt virus promoter S4 region or subterraneanclover stunt virus promoter S7 region comprising an Adh1 intron (WO9606932) a first nucleotide sequence comprising in order

a. the nucleotide sequence of SEQ ID No 7 from nucleotide 2314 tonucleotide 2514;

b. the nucleotide sequence of SEQ ID No 7 from nucleotide 429 tonucleotide 629;

C. the nucleotide sequence of SEQ ID No 7 from nucleotide 5052 tonucleotide 5270;

d. the nucleotide sequence of SEQ ID No 8; and

e. the nucleotide sequence of SEQ ID No 9;

a second nucleotide sequence which comprise the complement of the firstnucleotide sequence

a transcription termination and polyadenylation signal

The DNA molecule is introduced into barley plants, and BYDV/CYDVresistant barley plant lines are isolated.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 9 <210> SEQ ID NO 1 <211> LENGTH: 1594<212> TYPE: DNA <213> ORGANISM: Barley yellow dwarf virus <220> FEATURE:<221> NAME/KEY: misc_feature<223> OTHER INFORMATION: part of BYDV genome sp#anning ORF1 and 5′ end of       ORF2 <400> SEQUENCE: 1ttttcgaaat acttatagga gctagcgcta aggcggtcaa agacttcatc ag#ccattgtt     60actctcgttt gaaatctata tattattctt tcaaacgatg gctgatggag at#atctgggc    120agtttaaggc ccacgacgcc tttgtcaaca tgtgcttcgg gcacatggct ga#cattgagg    180acttcgaggc agagctcgct gaggagtttg ccgagaggga ggatgaggtg ga#agaggcaa    240ggagcctctt gaaactgctg gttgcccaaa aatctaaaac tggggtaacc ga#ggcttgga    300ccgacttctt tacgaagtcg agaggtggcg tctatgcgcc actttcctgc ga#gcctacca    360ggcaggagct agaagctaag agcgagaagc tcgaaaagct tctagaagaa ca#gcaccaat    420tcgaggtgcg agcggccaag aaatacatta aggaaaaagg ccgcggcttc at#caactgtt    480ggaacgactt gcgaagtcgt ctcaggctgg tgaaggacgt caaggacgag gc#gaaggaca    540acgccagagc tgctgccaag atcggagcag aaatgttcgc ccccgttgac gt#gcaagacc    600tctacagttt cacagaggtc aagaaggtgg agaccggcct catgaaggag gt#cgtgaaag    660agagaaacgg cgaagaagag aaacacctcg agcccatcat ggaagaggtg ag#atctatca    720aggacaccgc tgaagctagg gacgccgcct ccacttggat aacagagaca gt#taagctga    780agaattctac gcttaatgcc gatgaattgt ctctggccac catcgcccgc ta#cgtcgaaa    840acgtagggga caaattcaaa ctcgacattg ctagcaagac atatctaaag ca#agtcgcat    900cgatgtctgt accaattcca actaacaagg acatcaaatt gaagatggtg ct#gcagagtc    960ctgaagcacg tgccaggcgg gaaccgttgg acgtgcttga ctctgtgggt tt#ttagaggg   1020gctctgtacc gcctctggtt ttgagagccc attccctatt ctcgggctgc ca#gagattgc   1080ggtcacagac ggagcccggc tccgtaaggt tagtagtaat attagatacc tt#agccaaac   1140ccacctaggt cttgtatata aggcaccaaa tgcctccctg cacaatgcgc tt#gtagcagt   1200ggagagaaga gttttcacag taggaaaggg ggacaaagca atctaccccc cc#cgcccaga   1260gcatgacatt ttcactgata caatggatta cttccaaaaa tccattatag aa#gaggtggg   1320atactgtaga acatatccag cgcaactcct ggctaacagc tatagcgcag ga#aagagagc   1380catgtatcac aaagccattg catcattgag gactgtccct tatcatcaga ag#gatgctaa   1440tgtgcaagct ttcctgaaga aggaaaaaca ctggatgacc aaggacatcg cc#ccccgatt   1500gatttgcccc cgcagcaagc ggtacaacat catcctagga actcgtttga aa#ttcaacga   1560 gaagaagatc atgcacgcta tcgatagtgt gttc       #                   #      1594 <210> SEQ ID NO 2 <211> LENGTH: 986<212> TYPE: DNA <213> ORGANISM: Barley yellow dwarf virus <220> FEATURE:<221> NAME/KEY: misc_feature<223> OTHER INFORMATION: part of BYDV genome sp#anning the 3′ end of ORF2 <400> SEQUENCE: 2ggatccccca ctgtgctttc tggctatgac aactttaaac aaggaagaat ca#tagccaag     60aagtggcaaa agtttgcatg ccccgtcgcc atcggcgtgg atgctagccg ct#ttgaccaa    120cacgtgtcag agcaggcgct taagtgggaa cacgggatat acaatgggat ct#tcggagac    180agcgaactgg ctcttgcact tgaacatcaa atcaccaaca acatcaagat gt#ttgttgaa    240gataaaatgc ttaggttcaa ggtaaggggc cacagaatgt ccggtgacat ta#ataccagc    300atgggaaata agctcataat gtgcggcatg atgcatgcat atttcaagaa gc#tgggtgtt    360gaagctgaac tttgtaacaa cggagacgac tgtgtcatca tcactgatag ag#ccaatgag    420aagctctttg atggcatgta cgaccatttc ctccagtatg gcttcaacat gg#tgaccgaa    480aaaccagttt acgaactgga gcaattggag ttttgccagt caaaaccggt ct#ctattaat    540ggaaagtata gaatggtcag aaggcccgat agcataggca aagatagcac aa#cactactg    600agcatgctca atcaatccga cgtcaagagc tacatgtcgg ctgttgctca gt#gtggcctg    660gtgctcaacg ctggagtacc catacttgaa agtttctata aatgcctata ta#gaagctcg    720gggtacaaga aagtgagtga ggaatttatt aaaaacgtca tatcgtatgg aa#cagatgag    780agactacaag gtagacgtac ctataatgaa acacctatca caaaccacag ta#gaatgtcc    840tactgggaat cattcggagt tgaccctaag atacagcaaa tcgtcgagag gt#actacgac    900ggtcttacgg taagtgccca actccagagc gtgaaggtga cgactccaca tc#tgcaatca    960 atactacttt ccataccgga aaacca          #                   #             986 <210> SEQ ID NO 3 <211> LENGTH: 65<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer 1 <400> SEQUENCE: 3accattctat tgtgctctcg cacagagata agcaggaaac catggttttc ga#aatactaa     60 taggt                  #                  #                   #            65 <210> SEQ ID NO 4 <211> LENGTH: 28<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer 2 <400> SEQUENCE: 4ccggaattct taatattcgt tttgtgag          #                  #             28 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer 3 <400> SEQUENCE: 5tgtggcagtg gagagaagag             #                  #                   # 20 <210> SEQ ID NO 6 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer 4 <400> SEQUENCE: 6atgttgttgg tgatttggtg             #                  #                   # 20 <210> SEQ ID NO 7 <211> LENGTH: 5677<212> TYPE: DNA <213> ORGANISM: Barley yellow dwarf virus <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1)<223> OTHER INFORMATION: “n” = a,g,c,or t <300> PUBLICATION INFORMATION:<308> DATABASE ACCESSION NUMBER: Genbank/X07653<309> DATABASE ENTRY DATE: 1995-06-12<313> RELEVANT RESIDUES: (1)..(5677) <400> SEQUENCE: 7ngtgaagatt gaccatctca caaaagctgt tacgtgcttg taacacacta cg#cgcccgtt     60ttgtattcgg gaagtagttg cgaaaacggt ccccttattg cctgacaagc ta#agggccac    120ccttctttcc ccaccgccat catgtttttc gaaatactaa taggtgctag cg#ccaaggcg    180gtcaaagact tcattagcca ttgctattct agattgaaat ctatatacta tt#ctttcaag    240cgatggctaa tggagatatc agggcaattt aaggcccacg acgcctttgt ca#acatgtgc    300tttgggcaca tggctgacat tgaggacttc gaggcggaac tcgctgagga gt#tcgccgag    360agggaggatg aggtggaaga ggcgaggagc ctcttgaaac tgctggtcgc cc#aaaaatct    420aaatctgggg tgaccgaggc ttggaccgac ttttttacaa agtcgagagg tg#gtgtttac    480gcaccacttt cctgcgagcc taccaggcag gagctagaag tcaagagtga ga#aactcgag    540cgacttctag aagagcagca ccaatttgag gtgcgagcgg ccaagaaata ca#tcaaggaa    600aagggccgcg gcttcatcaa ctgctggaac gacttgcgga gtcgtctcag gt#tggtgaag    660gacgtcaagg acgaggcgaa ggacaacgcc agagctgctg ccaagattgg ag#cagaaatg    720ttcgcccctg ttgacgtgca ggacctctac agtttcacgg aggtcaagaa gg#tggagacc    780ggcctcatga aggaggtcgt gaaagagaaa aacggcgaag aagagaaaca cc#tcgaaccc    840atcatggaag aggtgaggtc catcaaggac accgccgaag ccagggacgc cg#cctccact    900tggataacag agacagttaa gctgaagaac gcaacgctta acgcagatga ac#tgtctctt    960gccaccatcg cccgctacgt tgaaaacgta ggggacaagt tcaaactcga ca#ttgctagt   1020aaaacatatc taaagcaagt cgcatcgatg tctgtaccaa ttccaaccaa ca#aagacatc   1080aaattgaaga tggtgctaca gagtcctgaa gcacgtgcca ggcgggaacg ca#tggacgtg   1140cttgactctg tgggttttta gaggggctct gtaccgcctc tggttttgag ag#cccattcc   1200ctattctcgg gctgccagag attgcggtca cagacggagc ccggctccgc aa#ggttagta   1260gcaatattag ataccttagc caaacccatc taggtcttgt atataaggca cc#aaatgcct   1320ccctgcacaa cgcgcttgtg gcagtggaga gaagagtttt tacagtagga aa#gggggaca   1380aggcaatcta ccccccccgc cctgagcatg acattttcac tgatacgatg ga#ttactttc   1440aaaaatccat tatagaagag gtgggatact gtaaaacata tccagcgcaa ct#cctggcta   1500atagctatag cgcaggaaag agggccatgt atcacaaagc cattgcatcc tt#gaaaactg   1560tcccatatca tcagaaggat gccaatgtgc aagctttcct gaagaaggaa aa#acattgga   1620tgaccaagga catcgccccc cgattgattt gcccccgcag caagcggtat aa#tatcatcc   1680taggaactcg tttgaaattc aacgagaaga agatcatgca cgccatcgat ag#tgtgtttg   1740gatcccccac tgtgctttct ggttatgaca acttcaaaca aggaagaatc at#agccaaaa   1800agtggcaaaa gtttgcatgc cccgtcgcca tcggcgtgga tgctagccgc tt#tgaccaac   1860atgtgtcaga gcaggcgctt aagtgggaac acgggatata caatggaatc tt#cggagaca   1920gcgaaatggc tcttgcactt gaacaccaaa tcaccaacaa catcaagatg tt#tgttgaag   1980acaaaatgct taggttcaag gtaagaggcc acagaatgtc cggtgacatt aa#taccagca   2040tgggaaataa gctcataatg tgcggcatga tgcacgcata tcttaagaag ct#gggtgttg   2100aagctgaact atgtaataac ggagacgact gtgtcatcat cactgataga gc#caatgaga   2160agctctttga tggcatgtac gaccatttcc tccagtatgg cttcaacatg gt#gaccgaaa   2220aaccagttta cgaactggaa caattggagt tttgccagtc aaaaccggtc tc#tattaatg   2280gaaagtatag aatggtcaga aggcccgata gcataggcaa agatagcaca ac#actactga   2340gcatgctcaa tcaatccgac gtcaagagct acatgtcggc tgtcgctcag tg#tggtttgg   2400tgctcaacgc tggagtaccc atacttgaaa gtttctataa atgcctatat ag#gagctcgg   2460ggtacaaaaa agtgagtgag gaatttatta aaaacgtcat atcgtatgga ac#agatgaga   2520gactacaagg tagacgtacc tataatgaaa cacctatcac aaaccacagt ag#aatgtcct   2580actgggaatc attcggagtt gaccctaaga tacagcaaat cgtcgagagg ta#ctacgacg   2640gtcttacggt aagtgcccaa ctccagagtg tgaaggtgac gactccacat ct#gcaatcaa   2700tactgctttc cataccggaa aaccactcac aaaacgaata ttaattacca aa#tcttagct   2760gggtttggga tagggtttat agttagtata ccctgtacat tagctctcgc gt#actttatt   2820tacaataaag tttcagacac cactagagag gtggtgaatg aattcagtag gt#cgtagagg   2880acctagacgc gcaaatcaaa atggcacaag aaggaggcgc cgtagaacag tt#cggccagt   2940ggttgtggtc caacccaatc gagcaggacc cagacgacga aatggtcgac gc#aagggaag   3000aggaggggca aattttgtat ttagaccaac aggcgggact gaggtattcg ta#ttctcagt   3060tgacaacctt aaagccaact cctccggggc aatcaaattc ggccccagtc ta#tcgcaatg   3120cccagcgctt tcagacggaa tactcaagtc ctaccatcgt tacaagatca ca#agtatccg   3180agttgagttt aagtcacacg cgtccgccaa tacggcaggc gctatcttta tt#gagctcga   3240caccgcgtgc aagcaatcag ccctgggtag ctacattaat tccttcacca tc#agcaagac   3300cgcctccaag accttccggt cagaggcaat taatgggaag gaattccagg aa#tcaacgat   3360agaccaattt tggatgctct acaaggccaa tggaactacc actgacacgg ca#ggacaatt   3420tatcattacg atgagtgtca gtttgatgac ggccaaatag gtagactcct ca#acaccgga   3480accaaaacct gcaccggaac caacaccaac cccccagcca acgccggctc ca#cagcccac   3540acctgaacca actcctgcac ctgtccccaa aagattcttc gagtatatcg ga#actcctac   3600cggtacaatc tcgactagag agaacactga cagtatatct gtcagcaagc tc#ggtggaca   3660gtcgatgcag tacattgaga atgagaaatg tgaaacgaaa gtcatcgatt cc#ttttggag   3720cactaacaac aacgtttctg cgcaagcagc tttcgtttat ccagtgccag ag#ggatcata   3780cagcgttaac atttcgtgcg aaggcttcca gtcagttgac cacatcggtg gc#aacgagga   3840cggctattgg attggtttaa ttgcctactc caattcgtct ggcgataatt gg#ggagttgg   3900caattacaaa gggtgcagtt ttaagaattt cttggcaacc aacacttgga ga#ccaggcca   3960caaagatctc aagttgactg attgccagtt cacagatgga caaatagttg aa#agggacgc   4020cgtgatgtct ttccacgtag aagcaacagg caaggatgcc agcttctacc tc#atggctcc   4080caaaacaatg aaaactgaca aatacaacta tgttgtctca tatggagggt ac#acaaacaa   4140gcgaatggaa ttcggtacca tatctgtgac atgtgatgaa tccgatgttg ag#gcagaacg   4200aataacaagg cacgctgaaa cgcccatacg ttctaaacat attcttgttt ct#gagcggta   4260tgcggaacca ttgcccacca tagtcaacca aggcttgtgt gatgtgaaaa ct#cccgagca   4320agaacaaaca ctggtggatg aagatgacag acaaactgtt tctactgaat ct#gatatagc   4380actcctggag tatgaggctg caacagctga gattccggat gctgaagagg ac#gttttgcc   4440ctccaaggaa cagttgtctt caaaaccaat ggatacgtct ggcaatataa ta#ccaaaacc   4500caaggaacct gaagtacttg ggacatacca aggacagaac atttatcctg aa#gacgtacc   4560tccaatggcg cggcagaaat tgagagaagc cgcgaatgcg ccttccacgc ta#ctctatga   4620aagaagaacc ccaaagaaga gtggcaactt tttatccaga cttgtagaag cg#aataggtc   4680ccctactact cccactgccc catccgtgtc aactacttca aacatgacaa gg#gagcagct   4740ccgggagtac actaggatta gaaattccag cggaatcaca gcagcaaagg cg#tacaaggc   4800gcaattccag tgaagacaac accactagca caaatcggat cctgggaaac ag#gcagaact   4860tcggttcgta agctcgggta ggccgtcaac ctaccgccgt atcgtattgt gt#ttggccga   4920tggaggatct tcacgttatc gccgtttgta ttcttgcttt gactgtgctc tc#tggggtag   4980gcgctgtttt gagttgctgc cgttggtgct gcagcaatcc ttttcctccc tc#cctctctt   5040ctgttcaagc aaaagactct cgatctgtgc gagagacaat caaaaatatc ga#gggagctt   5100cggctcagtg aggggattaa cgacccccag taatggccgg tcctggcgga ca#taaataac   5160ccgctatagg acgaagtggt agccaccact gatcaaatgg caaacatgct tc#tgtgttgt   5220acactgcccc ggagcctacc gggtcaacaa ggctatccca ccaacccgat ga#aatgaggg   5280tggagtgagc ggagtgggtg acttcgtgat gtacacccga tcgtcaggat tg#aagacgtt   5340aaaactcgac gacctggtac aagtcgttaa actgactcgg gtggatacac ca#cacccggc   5400ccagcatgtt ggcataccca cgatacgaaa cgtgggtctc ttggagccac ta#cctgtgat   5460gcaaggtagg gtatgagtct tagcaagctc tgagccagga gatggacata aa#ccatagca   5520atccaacgtg taaccgcaat ggggcaaaca acaggtgaac cgtgtccacg gg#cctggtta   5580ccgaaaggaa agccagtatc caacacagca atgtgttggg ggtcacacct tc#ggggtact   5640 cttaacgctg acactcgaaa gagcagttcg gcaaccc      #                   #    5677 <210> SEQ ID NO 8 <211> LENGTH: 196<212> TYPE: DNA <213> ORGANISM: Cereal Yellow Dwarf Virus<400> SEQUENCE: 8ccgaggacca agtcgaaagt tttatcaatg tcctcgcggg taccgctggt gc#ccgtcctg     60atgagatctg cgacaagtgg cgcgatctct tggttccaac ggactgctct gg#tttcgact    120ggtcagtctc cgattggatg ctcgcagacg atatggaggt aagaaatcct ct#taccatcg    180 actgcaatga gctcac              #                  #                   #   196 <210> SEQ ID NO 9 <211> LENGTH: 195<212> TYPE: DNA <213> ORGANISM: Cereal Yellow Dwarf Virus<400> SEQUENCE: 9gttcacacat cttcaagtct cctaccctcg ccattccggt caatgccaac aa#gatattgt     60accgcttgat ccacgggtac aatccggaat gtggaaacgc tgaggtgatt gt#caattacc    120tcaatgcagc cagctcagtg ctgcatgagc tccgtcatga tcaggagctt tg#cgcgttat    180 tgcatatgtg gttag               #                  #                   #   195

What is claimed is:
 1. A non-naturally occurring DNA molecule comprisinga plant-expressible promoter, a transcription termination andpolyadenylation signal; and a DNA region encoding a first nucleotidesequence of at least 19 nucleotides wherein said first nucleotidesequence has 100% nucleotide sequence identity to a sequence in thesense nucleotide sequence encoding an RNA dependent RNA polymerase of aBYDV isolate; and a second nucleotide sequence that has 100% nucleotidesequence identity to the complement of said first nucleotide sequence;wherein, when said DNA region is transcribed in the cells of a cerealplant, said first and second nucleotide sequences hybridize to form adouble stranded RNA.
 2. The DNA molecule of claim 1, wherein said sensenucleotide sequence is encoded by the nucleotide sequence of SEQ ID NO:1 from the nucleotide at position 1 to the nucleotide at position 1014.3. The DNA molecule of claim 1, wherein said sense nucleotide sequenceis encoded by the nucleotide sequence of SEQ ID NO: 1 and said secondnucleotide sequence is encoded by the complement of the nucleotidesequence of SEQ ID NO:
 1. 4. The DNA molecule of claim 1, wherein saidDNA region when transcribed in the cells of a cereal plant yields an RNAmolecule comprising a nucleotide spacer between said first nucleotidesequence and said second nucleotide sequence.
 5. The DNA molecule ofclaim 4, wherein said spacer is encoded by the nucleotide sequence ofSEQ ID NO:
 2. 6. A non-naturally occurring DNA molecule comprising aplant-expressible promoter, a transcription termination andpolyadenylation signal; and a DNA region encoding: a first nucleotidesequence of at least 19 nucleotides wherein said first nucleotidesequence has 100% nucleotide sequence identity to a sequence in thesense nucleotide sequence encoding an RNA dependent RNA polymerase of aBYDV isolate, and comprising at least one and maximally 10 additionalnucleotide sequences, each of said additional nucleotide sequenceshaving at least 19 nucleotides wherein each additional nucleotidesequence has 100% sequence identity to a sequence in the sensenucleotide sequence of a BYDV isolate genomic sequence or a CYDV isolategenomic sequence; and a second nucleotide sequence complementary to saidfirst nucleotide sequence; wherein, when said DNA region is transcribedin the cells of a cereal plant, said first and second nucleotidesequences hybridize to form a double stranded RNA.
 7. The DNA moleculeof claim 6, wherein said DNA region comprises in order: a nucleic acidcomprising the nucleotide sequence of SEQ ID No 7 from the nucleotide atposition 2314 to the nucleotide at position 2514; a nucleic acidcomprising the nucleotide sequence of SEQ ID No 7 from the nucleotide atposition 429 to the nucleotide at position 629; a nucleic acidcomprising the nucleotide sequence of SEQ ID No 7 from the nucleotide atposition 5052 to the nucleotide at position 5270; a nucleic acidcomprising the nucleotide sequence of SEQ ID No 8; and a nucleic acidcomprising the nucleotide sequence of SEQ ID No
 9. 8. A method forproducing a cereal plant resistant to a Barley Yellow Dwarf viruscomprising the steps of: producing a population of transgenic cerealplant lines comprising the DNA molecule of any one of claims 1 to 7integrated into the genome of the cells of transgenic plants of saidplant lines; and isolating a transgenic cereal plant line resistant toBarley Yellow Dwarf virus infection.
 9. The method of claim 8, whereinsaid cereal plant is selected from the group of wheat, barley and oats.10. The method of claim 8, wherein said cereal plant is barley.
 11. Amethod for producing a cereal plant resistant to a Barley Yellow Dwarfvirus comprising the steps of producing a population of transgeniccereal plant lines comprising the DNA molecule of any one of claims 1 to7 integrated into the genome of the cells of transgenic plants of saidplant lines; isolating a transgenic cereal plant resistant to BarleyYellow Dwarf virus infection; and crossing the isolated transgeniccereal plant resistant to Barley Yellow Dwarf virus infection withanother cereal plant to obtain progeny plants comprising the DNAmolecule of any of claims 1 to
 7. 12. A method for producing a cerealplant resistant to a Barley Yellow Dwarf virus in the presence ofco-infecting virus, comprising the steps of: producing a population oftransgenic cereal plant lines comprising the DNA molecule of any one ofclaims 1 to 7 integrated into the genome of the cells of transgenicplants of said plant lines; and isolating a transgenic cereal plant thatis resistant to Barley Yellow Dwarf virus infection in the presence ofsaid co-infecting virus.
 13. The method of claim 12, wherein saidco-infecting virus is Cereal Yellow Dwarf virus.
 14. A transgenic cerealplant comprising stably integrated into the genome of the cells of saidplant the DNA molecule according to any one of claims 1 to 7 and whereinsaid cereal plant is resistant to BYDV virus infection and replicationof said virus.
 15. The cereal plant of claim 14 wherein said plant isselected from the group consisting of wheat, barley, rye and oats. 16.The cereal plant of claim 15 wherein said plant is barley.
 17. Themethod of claim 9, wherein said cereal plant is wheat.
 18. The method ofclaim 9, wherein said cereal plant is oats.
 19. The cereal plant ofclaim 15 wherein said plant is wheat.
 20. The cereal plant of claim 15wherein said plant is oats.